毒死蜱、氧毒死蜱体外诱导人胚肝造血干细胞DNA损伤和MLL基因重组的特征和机制

Chlorpyrifos (CPF) is the organophosphate pesticide that is widely used in agriculture and at home. Maternal exposure to CPF has been associated with genetic abnormalities during fetal development and increased risk of infant acute leukemia (IAL) in offspring. The majority of patients with IAL acquire 11q23 rearrangement of the mixed lineage leukemia gene (MLL+) in utero. While the mechanistic basis for MLL translocations is currently unknown, the involvement of topoisomerase II (Topo II) inhibition and/or oxidative stress mediated DNA damage and chromatin fragmentation during early apoptosis have been suggested. We have previously observed that CPF and its toxic metabolite, chlorpyrifos oxon (CPO), can induce DNA injury, early apoptosis and MLL translocations in human fetal CD34+HSC. In the current project, we will utilize human fetal CD34+HSC to explore the characteristics of CPF/CPO induced MLL rearrangements, including types of rearrangements, specific lineages within HSC populations that are sensitive to MLL rearrangements, and whether a subset of MLL rearranged cells undergo clonal expansion and proliferate in culture. Furthermore, we will carefully examine the role of Topo II inhibition and oxidative stress in CPF/CPO induced MLL rearrangements. Our findings may help provide scientific evidence for therapeutic intervention and disease prevention.

毒死蜱(CPF)是一种广泛使用的有机磷杀虫剂，其低水平长期暴露对人体产生的效应，特别是孕期暴露造成胎儿和出生后儿童发育的影响已广受关注。流行病研究显示，孕期暴露CPF可增加婴儿和儿童急性白血病发病的风险。MLL基因重组是婴儿急性白血病主要病理特征，可能与Topo II抑制及/或氧化应激诱导的DNA氧化损伤和细胞早期凋亡有关。我们前期研究显示，CPF及其体内代谢产物－氧毒死蜱(CPO)在体外可诱导DNA损伤、细胞早期凋亡和MLL重组，但机制尚不清楚。本课题拟以原代人胚肝CD34+造血干细胞(HSC)为体外模型，探讨CPF、CPO体外诱导人胚肝HSC的MLL重组特点，包括重组类型，敏感靶细胞，及MLL+HSC的分裂增殖特征等，并确定Topo II抑制和氧化应激在CPF、CPO诱导MLL重组的作用，其结果将为孕期暴露环境毒物和药物诱导的婴儿和儿童急性白血病的病因学及发病机制提供实验依据。

毒死蜱（CPF）是一种广泛使用的有机磷杀虫剂，其低水平长期暴露对人体产生的效应，特别是孕期暴露造成胎儿和出生后儿童发育的影响已广受关注。流行病研究显示，孕期暴露CPF等杀虫剂可增加婴儿和儿童急性白血病发病的风险。MLL基因重组是婴儿急性白血病主要病理特征，可能与Topo II抑制及/或氧化应激诱导的DNA氧化损伤和细胞早期凋亡有关。本课题以原代人胚肝CD34+造血干细胞（CD34+HSC）为体外生物模型，运用前沿的细胞分子生物学技术方法探讨CPF、CPO体外诱导人胚肝HSC的MLL重组特点，并确定Topo II抑制和氧化应激在CPF、CPO诱导MLL重组的作用。包括：联合荧光原位杂交（FISH）和多重逆转录聚合酶链反应确定CPF、CPO诱导的MLL基因重组和类型；荧光激活细胞分选术确定MLL基因重组特异的敏感干细胞亚系；流式细胞仪确定携带MLL基因重组细胞在体外克隆扩增；Comet确定DNA损伤性质；生化技术和流式细胞仪确定Topo II和氧化损伤在MLL基因重组的角色；基因芯片（Human Genome U133 Plus2.0）技术分析CPF、CPO诱导的人胚肝HSC的基因表达谱，并筛选出与儿童急性白血病相关的基因。其结果将为孕期暴露环境毒物和药物诱导的婴儿和儿童急性白血病的病因学及发病机制实验依据。